Preparation and use of photopolymerized microparticles

ABSTRACT

Methods of forming crosslinked polymer particles in situ from polymer precursors such as monomers or oligomers, comprising exposing a composition comprising at least one polymer precursor, a solvent or solvent mixture, and an antisolvent or antisolvent mixture to photoradiation under conditions whereby particles are formed are provided. The polymer precursor may be photosensitive, or a separate polymerization initiator may be used. In a preferred embodiment, the polymer precursor is insoluble in the antisolvent or antisolvent mixture and the solvent or solvent mixture is soluble in the antisolvent or antisolvent mixture at the concentrations used. Crosslinked polymer particles and crosslinked polymer particles comprising a polymer and a bioactive material are also provided. The polymer may be erodable, and the polymer particles formed may be used in a variety of applications, including controlled release of bioactive materials such as drugs. Polymer particles formed using the methods of the invention have low residual solvent levels and high additive encapsulation efficiencies. The processes of the invention allow control of particle size and morphology, use low operating temperatures and are useful for efficient bulk production.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.10,161,544, filed Jun. 3, 2002, now allowed, which is acontinuation-in-part of U.S. patent application Ser. No. 09/451,481,filed Nov. 30, 1999, now U.S. Pat. No. 6,403,672, which claims thebenefit of U.S. Provisional Patent Application No. 60/110,816, filedNov. 30, 1998, which are hereby incorporated in their entirety byreference to the extent not inconsistent with the disclosure herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support by the NationalInstitutes of Health under grant number 5 R01 HL59400 and the NationalScience Foundation. The federal government may have certain rights inthis invention.

BACKGROUND OF THE INVENTION

This invention relates generally to polymer particles and methods ofmaking and using the same.

Small (micron- and nano-sized) polymer particles are useful for manyapplications, including pharmaceutical uses. Polymer microparticles areuseful for injectable and implantable devices because they have a longcirculation time in the body and are efficient drug, enzyme, and proteincarriers (Tom, J. W. et al. (1993), “Applications of SupercriticalFluids in the Controlled Release of Drugs,” in Supercritical FluidEngineering Science, pp. 238-257). Crosslinked polymer microparticleshave material property benefits over linear polymer particles includingimproved mechanical strength, greater control of transport properties,material property adjustability and dimensional stability. Someapplications of crosslinked polymers are listed in Cooper, A. L. andHolmes, A. B. (1998) Proceedings of the 5^(th) Meeting of SupercriticalFluids Materials and Natural Products Processing, pp. 843-848. Polymermicroparticles (both linear and crosslinked) have been used inapplications such as dental composites, biostructural fillers andcontrolled release devices. Some applications of synthetic bonecomposites are listed in Popov, V. K. et al. (1998) Proceedings of the5^(th) Meeting of Supercritical Fluids Materials and Natural ProductsProcessing, pp. 45-50.

Controlled release devices are useful in many applications, from medicalto agricultural purposes. (Langer, R. (1993), Polymer-Controlled DrugDelivery Systems,” Acc. Chem. Res. 26:537-542; U.S. Pat. No. 5,043,280).Controlled release delivery systems for drugs have a wide variety ofadvantages over conventional forms of drug administration. Some of theseadvantages include: decreasing or eliminating the oscillating drugconcentrations found with multiple drug administrations; allowing thepossibility of localized delivery of the drug to a desired part of thebody; preserving the efficacy of fragile drugs; reducing the need forpatient follow-up care; increasing patient comfort; and improvingpatient compliance. (Langer, R. (1990), “New Methods of Drug Delivery,”Science 249:1527-1533).

Crosslinked polymeric release devices have the capability to modify therelease profile of a drug or other chemical by modifying the structureof the crosslinked polymer network. A crosslinked polymer network canprovide diffusion controlled release of a drug or other chemical. Therate of diffusion of the drug or other bioactive material to be releasedcan be influenced by the mesh size of the network, or the distancebetween crosslinks, which depends upon the extent of crosslinking in thenetwork. In a biodegradable system, the mesh size of the network willincrease with time as the network degrades.

Current polymer microparticle manufacturing techniques all suffer fromone or more disadvantages. For example, the spray drying techniqueusually requires evaporation of solvent in hot air. The hightemperatures used can degrade sensitive drugs and polymers. In thermalpolymerization, monomer is heated to induce polymerization. Again, thehigh temperatures used can cause degradation (including lowering theactivity of biologically active substances).

Emulsion and suspension polymerizations (see, for example, U.S. Pat. No.5,603,960 (O'Hagan., et al.)) involve combinations of solvents,emulsifiers, and surfactants where dispersed islands of monomerpolymerize through chemical reaction in a sea of solvent. These methodsoften involve operation at high temperatures and thus have the problemsdiscussed above, use large volumes of solutions that are oftenenvironmentally unfriendly, and permit only minimal control overparticle size and morphology.

A number of different techniques have been developed to form smallparticles of polymers using the solvent power of supercritical fluids.Supercritical fluids have liquid-like densities, very largecompressibilities, viscosities between those of liquids and gases, anddiffusion coefficients that are higher than liquids. Due to the highcompressibility, the density (and solvent power) of a supercriticalfluid can be adjusted between gas- and liquid-like extremes withmoderate changes in pressure (Debenedetti, P. G. et al. (1993), “RapidExpansion of Supercritical Solutions (RESS): Fundamentals andApplications,” Fluid Phase Equilibria 82:311-321).

The Rapid Expansion of Supercritical Solution (RESS) technique has beenused to form small particles of poly(L-lactic acid) (Debenedetti, P. G.et al. (1993), “Supercritical Fluids: A New Medium for the Formation ofParticles of Biomedical Interest,” Proceed. Intern. Symp. Control Rel.Bioact. Mater. 20:141-142) and particles of poly(DL-lactic acid) withembedded lovastatin (Tom, J. W. et al. (1993), “Applications ofSupercritical Fluids in the Controlled Release of Drugs,” inSupercritical Fluid Engineering Science, pp. 238-257). In the RESStechnique, particles of polymer may be made when a polymer is dissolvedin a supercritical fluid (usually carbon dioxide) followed by rapidexpansion of the fluid. This technique is limited in applicability tocompounds that are soluble in the supercritical fluid. Since most drugsare not soluble in supercritical fluids and most polymers have very lowsolubility in supercritical fluids, the RESS process is not broadlyapplicable for drug encapsulation (McHugh, M. and Krukonis, V. (1994)Supercritical Fluid Extraction, Butterworth-Heinemann).

In the Precipitation by a Compressed Antisolvent (PCA) technique (alsoknown as the Gas Antisolvent technique), a solid of interest isdissolved in a solvent and the resulting solution is sprayed into acompressed antisolvent (see, for example, U.S. Pat. Nos. 5,833,891 and5,874,029). In this technique, the antisolvent and solvent are soluble,but the solid of interest is not soluble in the antisolvent. Theantisolvent is believed to extract the solvent, precipitating particlesof the solid of interest (Randolph, T. W. et al. (1993) Biotech. Prog.9:429-435). Microparticles of insulin have reportedly been formed usingthis technique (Yeo, S. D. et al. (1993), “Formation of MicroparticulateProtein Powders Using a Supercritical Fluid Antisolvent,” Biotech.Bioeng. 41:341-346) and linear polymer microparticles have been formedusing polymer starting materials (Bodmeier, R. et al. (1995), “PolymericMicrospheres Prepared by Spraying into Compressed Carbon Dioxide,”Pharm. Res. 12 (8):1211-1217; U.S. Pat. Nos. 5,833,891; 5,874,029).

There is a need for polymer particles with low residual solvent levels,high additive encapsulation efficiencies, and processes of makingpolymer particles that allow control of particle size and morphology,with low operating temperatures and efficient bulk productioncapability. Formation of polymer particles with degradable networks,whether by surface or bulk degradation, are also needed for controlledrelease of drugs, for example. In particular, highly crosslinked polymernetworks with degradable chemistries are desired. Preferably, the extentof crosslinking or mesh size of such highly crosslinked polymer networksis controlled to tailor the release profile of the drug or otherchemical to be released. In addition, there is a need for a process thatproduces polymer particles in situ from polymer precursors such asmonomers or oligomers.

BRIEF SUMMARY OF THE INVENTION

In a general description of the invention, a method of forming polymerparticles comprising exposing a composition comprising at least onepolymer precursor, a solvent or solvent mixture, and an antisolvent orantisolvent mixture to photoradiation under conditions whereby particlesare formed is provided. If the precursor is not photosensitive, at leastone photoinitiator is present in the composition. The solvent is notrequired if the polymer precursor is liquid or liquifiable. If used, thesolvent is chosen so that the polymer precursor is soluble in thesolvent at the concentrations used, and the antisolvent and solvent aresoluble in each other at the concentrations used. The polymer precursoris preferably insoluble in the antisolvent, but as long as nucleationand particle formation occur, any solubility condition may be present.Bioactive materials such as drugs may also be included in thecomposition.

Also provided is a method of forming polymer particles from a solutioncomprising contacting a solvent or solvent mixture and at least onepolymer precursor with an antisolvent or antisolvent mixture underconditions whereby particles are generated; and exposing said particlesto photoradiation, whereby polymer particles are formed. Preferably thepolymer precursor is insoluble in the antisolvent or antisolventmixture.

Also provided are polymer particles prepared by the methods of theinvention that are between about 0.001 μm to about 200 μm in diameter.Each individual particle size and all intermediate ranges of particlesize are included in the invention In one embodiment, particles areprovided that are between about 0.1 μm and about 100 μm in diameter.

Linear and crosslinked polymer particles may be formed using the methodsof the invention. Crosslinked polymer particles in which the crosslinkedpolymer forms a network are also provided. The mesh size of the networkcan be between about 10 Angstroms and about 500 Angstroms. Eachindividual mesh size and all the intermediate ranges of mesh sizes areincluded in the invention. For example, the mesh size can also beselected to be between about 10 Angstroms and about 100 Angstroms.Crosslinked particles comprising a multiplicity of convertedcarbon-carbon double bond functional groups are provided, wherein theconversion of the carbon-carbon double bonds is a measure of the extentof crosslinking. In one embodiment, the carbon-carbon double bondconversion in the particles is between about 20% and about 100%. Eachindividual value of carbon-carbon double bond conversion and all theintermediate ranges of carbon-carbon double bond conversion are includedin the invention. For example, the carbon-carbon double bond conversioncan be greater than about 70%.

Also provided is a method of forming polymer particles comprising:substantially dissolving at least one polymer precursor in a solvent orsolvent mixture to form a solution; contacting said solution with anantisolvent or antisolvent mixture in which said polymer precursor isinsoluble to form a composition comprising said precursor, and asubstantially soluble mixture of said solvent or solvent mixture andsaid antisolvent or antisolvent mixture; and exposing said compositionto sufficient photoradiation to initiate polymerization whereby polymerparticles are formed.

Also provided is a method of forming polymer particles comprising:establishing a flow of antisolvent in an optically accessible chamber;contacting a solution comprising at least one polymer precursor and atleast one polymerization initiator dissolved in a solvent or solventmixture with said antisolvent under conditions whereby particles areformed; and exposing said particles to photoradiation whereby polymerparticles are formed.

Also provided is a method for making crosslinked polymer particles witha desired double bond conversion amount comprising the steps of:exposing a composition comprising a polymer precursor, a non-aqueoussolvent or solvent mixture, and an antisolvent or antisolvent mixture tophotoradiation under conditions whereby crosslinked particles of thedesired conversion amount are formed, wherein the antisolvent is asupercritical or near supercritical fluid in which the polymer precursoris not substantially soluble.

Also provided is a method for making crosslinked polymer particleshaving a desired network mesh size comprising the steps of:

-   -   selecting a polymer precursor;    -   determining a double bond conversion amount which corresponds to        the desired network mesh size for the polymer;    -   exposing a composition comprising the polymer precursor, a        non-aqueous solvent or solvent mixture, and an antisolvent or        antisolvent mixture to photoradiation under conditions whereby        crosslinked particles having the double bond conversion amount        are formed, wherein the antisolvent is a supercritical or near        supercritical fluid in which the polymer precursor is not        substantially soluble and whereby the crosslinked particles have        the desired network mesh size.

Also provided is a method of forming copolymers comprising: dissolvingor suspending at least two polymer precursors or at least one polymerprecursor and at least one polymer in a solvent or solvent mixture toform a solution; contacting said solution with an antisolvent orantisolvent mixture to form a composition comprising: said precursors orsaid precursor and polymer; a soluble mixture of said solvent or solventmixture and said antisolvent or antisolvent mixture; and exposing saidcomposition to photoradiation whereby copolymer particles are formed.

Copolymers may also be formed where at least one polymer precursor or atleast one polymer are present in a solvent or solvent mixture, and atleast one polymer precursor or at least one polymer are present in theantisolvent or antisolvent mixture, providing that at least one polymerprecursor is present.

Also provided is a method of forming particles comprising a bioactivematerial and a polymer comprising: exposing a composition comprising atleast one bioactive material, at least one polymer precursor and anantisolvent or antisolvent mixture to photoradiation under conditionswhereby particles are formed.

Polymers formed may be erodable or nonerodable, biodegradable ornonbio-degradable and biocompatible or nonbiocompatible. Polymerparticles formed using the methods of the invention may be used forcontrolled release of a desired substance in an organism or system.Provided is a method of controlled release of a desired substancecomprising: preparing polymer particles that comprise a degradablepolymer and a desired substance; and exposing said polymer particles toconditions under which the polymer is degraded.

Methods of forming degradable particles comprising a degradable polymerand a pharmaceutical product comprising: exposing a compositioncomprising a solvent or solvent mixture, at least one polymer precursorcapable of forming a degradable polymer, at least one pharmaceuticalproduct, and an antisolvent or antisolvent mixture to photoradiationwhereby polymer particles that contain a degradable polymer and apharmaceutical product are formed are provided.

A pharmaceutical composition comprising polymer particles produced bythe methods of the invention and a pharmaceutically acceptable carrierare also provided. Polymer particles comprising at least one bioactivematerial and at least one polymer are also provided.

Crosslinked polymer particles comprising a degradable polymer are alsoprovided. Biodegradable crosslinked polymer particles are also provided.Crosslinked polymer particles further comprising at least one bioactivematerial are also provided.

An apparatus is provided for producing polymer microparticles whichcomprises: a reaction chamber; at least one inlet into said reactionchamber through which an antisolvent or antisolvent mixture, at leastone polymer precursor insoluble in said antisolvent or antisolventmixture, and a solvent or solvent mixture soluble in said antisolvent orantisolvent mixture pass into said chamber; and a light source opticallyconnected to said chamber wherein during operation of the chamber saidpolymer precursor is polymerized. The apparatus may be used with aphotosensitive polymer precursor, or a polymerization initiator may beadded.

Advantages of this photopolymerization technique include morphologicalcontrol through polymerization rate, process conditions, and initiationlocation. Processing time remains short while processing temperaturesremain low. Low operating temperatures are important since manypotential encapsulation additives degrade at moderate temperatures. Inaddition, particles formed using the method of the invention do notrequire further processing, for example solvent removal, before use.

Further objects and advantages of this invention will be apparent from aconsideration of the drawings and description herein.

“Microparticles” as used herein means particles that are less than about100 μm in diameter. “Nanoparticles” are particles that are less thanabout 1 μm in diameter. Both microparticles, nanoparticles and particlesof other sizes may be produced by the methods of the invention bychanging process parameters and choice of materials. Methods of changingthe process parameters and materials are described herein, ordeterminable by one of ordinary skill in the art without undueexperimentation.

“Polymer precursor” means a molecule or portion thereof which can bepolymerized to form a polymer or copolymer. Polymer precursors includeany substance that contains an unsaturated moiety or other functionalitythat can be used in chain polymerization, or other moiety that may bepolymerized in other ways. Such precursors include monomers andoligomers. A “multifunctional monomer” is a monomer having two or moresites available for bonding to other molecules during polymerization.Preferred precursors include those that are capable of being polymerizedby photoradiation. One class of polymer precursors of the invention arethose that are insoluble in the antisolvent or antisolvent mixture.Another class of polymer precursors of this invention arephotosensitive. If a polymer precursor that polymerizes photochemicallyis used (photosensitive polymer precursor), a separate photoinitatordoes not need to be used. Examples of photosensitive polymer precursorsinclude tetramercaptopropionate and3,6,9,12-tetraoxatetradeca-1,13-diene. Another class of precursors thatmay be used are radically polymerizable precursors. Another class ofprecursors that may be used are ionically polymerizable precursors.Another class of precursors that are useful in the invention arecationic precursors.

Some examples of precursors that are useful in the invention includeethylene oxides (for example, PEO), ethylene glycols (for example, PEG),vinyl acetates (for example, PVA), vinyl pyrrolidones (for example,PVP), ethyloxazolines (for example, PEOX), amino acids, saccharides,proteins, anhydrides, vinyl ethers, amides, carbonates, phenylene oxides(for example, PPO), acetals, sulfones, phenylene sulfides (for example,PPS), esters, fluoropolymers, imides, amide-imides, etherimides,ionomers, aryletherketones, olefins, styrenes, vinyl chlorides,ethylenes, acrylates, methacrylates, amines, phenols, acids, nitriles,acrylamides, maleates, benzenes, epoxies, cinnamates, azoles, silanes,chlorides, epoxides, lactones and amides. A preferred group ofprecursors includes all the above precursors with the exception offluoropolymers.

Polymer precursors useful for producing crosslinked polymer particlesinclude multifunctional monomers such as: vinyl acetates (for example,PVA), vinyl pyrrolidones (for example, PVP), vinyl ethers, olefins,styrenes, vinyl chlorides, ethylenes, acrylates, methacrylates,nitriles, acrylamides, maleates, epoxies, epoxides, and lactones. If acrosslinking agent is involved, polymers useful for producingcrosslinked polymer particles include monomers such as: ethylene oxides(for example, PEO), ethylene glycols (for example, PEG), vinyl acetates(for example, PVA), vinyl pyrrolidones (for example, PVP),ethyloxazolines (for example, PEOX), amino acids, saccharides, proteins,anhydrides, vinyl ethers, carbonates, phenylene oxides (for example,PPO), acetals, sulfones, phenylene sulfides (for example, PPS), esters,fluoropolymers, imides, amide-imides, etherimides, ionomers,aryletherketones, olefins, styrenes, vinyl chlorides, ethylenes,acrylates, methacrylates, amines, phenols, acids, nitriles, acrylamides,maleates, benzenes, epoxies, cinnamates, azoles, silanes, chlorides,epoxides, lactones and amides. Crosslinking agents include reactivegroups having photocrosslinkable carbon-carbon double bonds attached tothe ends of the polymer precursor chains. Such a carbon-carbon doublebond can provide two sites available for bonding to other molecules.Suitable reactive groups having photocrosslinkable carbon-carbon doublebonds include, without limitation, acrylates, methacrylates, alkenes andalkynes. Polymers having such reactive groups attached to the polymerprecursor chains are termed “functionalized polymers”. Polymerprecursors useful for producing crosslinked polymer particles alsoinclude copolymers of the above monomers. Copolymers of the abovemonomers with degradable or erodable polymers may be used to obtaindegradable or erodable crosslinked particles.

As used herein, “polymer” includes copolymers. “Copolymers” are polymersformed of more than one polymer precursor. Polymers that can be formedusing the methods of this invention include those which are preparedfrom precursors that, in a preferred embodiment are soluble in a solventthat is soluble in an antisolvent and can be polymerized with lightinitiation. One class of polymers that may be prepared using the methodof this invention includes those that are degradable, preferablybiodegradable. Another class of polymers that may be prepared using themethod of this invention includes those that are not degradable. Anotherclass of polymers that may be prepared using the method of thisinvention includes those that comprise one or more degradable polymersand one or more nondegradable polymers. Another class of polymers thatmay be prepared using the method of this invention includespoly(lactides), poly(glycolides), and poly(lactide-co-glycolides). In apreferred embodiment of the invention, the polymers are degradable orerodable.

“Degradable or erodable polymers” are those that degrade upon exposureto some stimulus, including time. Degradable or erodable polymersinclude biodegradable polymers. Biodegradable polymers degrade in abiological system, or under conditions present in a biological system.Preferred biodegradable polymers degrade in an organism, preferably amammal, and most preferably human. Examples of biodegradable polymersinclude those having at least some repeating units representative of atleast one of the following: an alpha-hydroxycarboxylic acid, a cyclicdiester of an alpha-hydroxycarboxylic acid, a dioxanone, a lactone, acyclic carbonate, a cyclic oxalate, an epoxide, a glycol, andanhydrides. Preferred degradable or erodable polymers comprise at leastsome repeating units representative of polymerizing at least one oflactic acid, glycolic acid, lactide, glycolide, ethylene oxide andethylene glycol.

A class of polymers included in this invention are biocompatiblepolymers. One type of biocompatible polymers degrade to nontoxicproducts. Specific examples of biocompatible polymers that degrade tonontoxic products that do not need removal from biological systemsinclude poly(hydro acids), poly(L-lactic acid) or L-PLA, poly(D,L-lacticacid) or D,L-PLA, poly(glycolic acid) and copolymers thereof.Polyanhydrides have a history of biocompatibility and surfacedegradation characteristics (Langer, R. (1993) Acc. Chem. Res.26:537-542; Brem, H. et al. (1995) Lancet 345:1008-1012; Tamada, J. andLanger, R. J. (1992) J. Biomat Sci.-Polym. Ed. 3:315-353).

Another class of polymers that may be prepared using the method of thisinvention include particles that are a suitable size for injection oradministration orally or incorporated in a preparation suitable for oraladministration. For oral or injectable delivery, it is preferred thatmost particles are less than 50 microns in diameter. Another class ofparticles that may be prepared using the method of this inventioninclude those that are a suitable size for inhalation or pulmonarydelivery. For pulmonary delivery, it is preferred that greater thanabout 90 weight percent of all solid particles in an administeredpharmaceutical formulation are of a size smaller than about 10 micronsand more preferably at least about 90 weight percent are smaller thanabout 6 microns, and even more preferably at least about 90 percent ofall solid particles are from about 1 micron to about 6 microns.Particularly preferred for pulmonary delivery applications are particlesof from about 2 microns to about 5 microns in size. Other classes ofparticles of suitable size for various applications are included in themethods of the invention.

Solvents useful in the invention include those that dissolve someportion of a polymer precursor and are preferably at least partiallysoluble in the antisolvent used. Preferably the solvent is miscible inthe antisolvent or antisolvent mixture at the temperature and pressureof operation. Preferred solvents are not water. Some examples ofpreferred solvents include methylene chloride, methanol, toluene,propanol, ethanol, acetone, ethers, hexanes, heptane, tetrahydrofuran,methyl ethyl ketone, chloroform, carbon tetrachloride, butanone,dimethyl sulfoxide, isopropanol, ethyl acetate, methyl acetate,n-methylpyrrolidine, propylene carbonate, alkanes, and acetonitrile. Ifa liquid or liquidizable polymer precursor is used, a solvent is notnecessary. One solvent or a mixture of solvents may be used.

Photoinitiators that are useful in the invention include those that canbe activated with light and initiate polymerization of the polymerprecursor. Preferred initiators include azobisisobutyronitrile,peroxides, phenones, ethers, quinones, acids, formates. Cationicinitiators are also useful in the invention. Preferred cationicinitiators include aryldiazonium, diaryliodonium, and triarylsulfoniumsalts. Most preferred initiators include Rose Bengal (Aldrich), Darocur2959 (2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone, D2959,Ciba-Geigy), Irgacure 651 (2,2-dimethoxy-2-phenylacetophenone, 1651,DMPA, Ciba-Geigy), Irgacure 184 (1-hydroxycyclohexyl phenyl ketone,1184, Ciba-Geigy), Irgacure 907(2-methyl-1-[4-(methylthio)phenyl]-2-(4-morpholinyl)-1-propanone, 1907,Ciba-Geigy), Camphorquinone (CQ, Aldrich), isopropyl thioxanthone(quantacure ITX, Great Lakes Fine Chemicals LTD., Cheshire, England). CQis typically used in conjunction with an amine such as ethyl4-N,N-dimethylaminobenzoate (4EDMAB, Aldrich) or triethanolamine (TEA,Aldrich) to initiate polymerization.

The wavelengths and power of light useful to initiate polymerizationdepends on the initiator used or the wavelength (or wavelengths) willactivate the photosensitive precursor. A combination of photosensitiveprecursor(s) and photoinitiator(s) may be used. When Rose Bengal is usedas the initiator, a visible light source is preferably used. Light usedin the invention includes any wavelength and power capable of initiatingpolymerization. Preferred wavelengths of light include ultraviolet orvisible. Any suitable source may be used, including laser sources. Thesource may be broadband or narrowband, or a combination. The lightsource may provide continuous or pulsed light during the process.

Chamber windows made from various materials may be used in the method ofthis invention. In addition, a filter may used to block a wavelengthfrom reaching the chamber, or allow a selected wavelength or wavelengthsof light to reach the chamber. The chamber windows themselves may act asthis filter, or a separate filter or filters may be used in conjunctionwith the chamber windows.

In one embodiment, a broadband light source may be used, and byselecting chamber window compositions and/or filter combinations, theselected wavelength or wavelengths of light may pass through thechamber. Light of different selected wavelengths may pass through thesame chamber at various locations. This feature may be used to activatemore than one photoinitiator.

As used herein, “antisolvent” is a substance in which the polymerprecursor is substantially not soluble. It should be understood that itis possible that the antisolvent may be capable of dissolving someamount of the precursor without departing from the scope of the presentinvention. The antisolvent is, however, preferably incapable ofdissolving a significant portion of the precursor such that at least asignificant portion of precursor is, in effect, not soluble in theantisolvent. Preferably, the antisolvent precipitation is conductedunder thermodynamic conditions which are near critical or supercriticalrelative to the antisolvent fluid. The antisolvent preferably comprisesany suitable fluid for near critical or supercritical processing. Thesefluids include carbon dioxide, ammonia, nitrous oxide, methane, ethane,ethylene, propane, butane, pentane, benzene, methanol, ethanol,isopropanol, isobutanol, fluorocarbons (includingchlorotrifluoromethane, monofluoromethane, hexafluoraethane and1,1-difluoroethylene), toluene, pyridine, cyclohexane, m-cresol,decalin, cyclohexanol, o-xylene, tetralin, aniline, acetylene,chlorotrifluorosilane, xenon, sulfur hexafluoride, propane and others.Carbon dioxide, ethane and propane are preferred antisolvents. Mostpreferably, carbon dioxide is used as the antisolvent. One antisolvent,or a mixture of different antisolvents may be used.

As used herein, “supercritical or near supercritical fluid” means asubstance that is above its critical pressure and temperature or issubstantially near its critical pressure and temperature.

Components that are “contacted” with each other refers to two or morecomponents physically near each other. Components that are contactedwith each other are preferably in intimate contact with each other sothat they may react with each other or affect each other. Contact mayinclude emulsions or microemulsions.

A “bioactive” material is any substance which may be administered to anybiological system, such as an organism, preferably a human or animalhost, and causes some biological reaction. Bioactive materials includepharmaceutical substances, where the substance is administered normallyfor a curative or therapeutic purpose. The bioactive material maycomprise a protein or other polypeptide, an analgesic or anothermaterial. In one embodiment, the bioactive material has a molecularweight less than 1000 Da. Suitable bioactive material includes, withoutlimitation, tacrine, erythromycin, erythromycin estolate, anderythromycin ethylsuccinate.

A “polymer shell” may be a continuous coating of polymer over somesubstance, but the coating is not required to be continuous. The polymershell may have nonhomogeneous regions where there is no coating, orregions where the coating is thicker than in other areas. The polymershell may be composed of different materials. Preferably, the polymershell is a homogeneous coating with uniform thickness. “Encapsulated” isintended to indicate a substance, such as a bioactive material, ishomogeneously distributed throughout the polymer.

“Linear polymers” are those polymers that are composed of individualpolymer chains that do not have bonds connecting the chains.“Crosslinked polymers” are those polymers that have bonds betweenpolymer chains. Branched polymers are also included in the invention.

“Soluble” does not necessarily mean completely soluble. As long as someportion of one substance dissolves in another substance, the substancesare soluble in each other.

Likewise, “insoluble” does not necessarily mean that no amount of onesubstance will dissolve in another substance.

A “composition” of substances is not intended to mean the substances aresoluble or miscible with each other, or react with each other. A“composition” is merely intended to mean all listed substances arepresent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a photopolymerization system.

FIG. 2 is a schematic diagram of a continuous photopolymerizationsystem.

FIG. 3 is a UV-vis spectrum of the chamber window, Rose Bengal (3.8×10⁻⁶wt percent) in methylene chloride and Rose Bengal bis(triethylammonium)salt (0.001 wt percent) in methylene chloride.

FIG. 4 is a Fourier transform infrared (FTIR) spectra of methacrylatedsebacic anhydride oligomer and poly(methacrylated sebacic anhydride).

FIG. 5 is a scanning electron microscopy (SEM) photograph ofpoly(methacrylated sebacic anhydride) microparticles precipitated byspraying and photopolymerizing a 10 wt % MSA solution through a 100 μmcapillary nozzle into CO₂ at a temperature of 37° C. and pressure of 8.5MPa.

FIG. 6 shows cloud point data for various molar concentrations(consistent with general operating conditions) of MSA monomer inmethylene chloride at 37° C.).

FIG. 7 is an SEM micrograph of PMSA precipitated by spraying andphotopolymerizing a 5 wt % MSA solution through an ultrasonic atomizingnozzle into CO₂ at a temperature of 25° C. and pressure of 8.5 MPa.

FIG. 8A-8C are SEM micrographs for 5% (A), 10% (B) and 20% (C) MSA.

FIGS. 9A and 9B are SEM micrographs showing triacrylate polymers undertwo experimental conditions.

FIGS. 10A and 10B are SEM micrographs showing triacrylate polymers undertwo experimental conditions.

FIG. 11 is a SEM micrograph for 5% MSA/5% PLA copolymer.

FIG. 12 is a fluorescence micrograph of PMSA particles encapsulated withtacrine.

FIG. 13 shows release data from PMSA microparticles encapsulated withtacrine.

FIG. 14 shows release data from particles containing Rose Bengalincorporated in a MSA matrix and polymerized into disks and release datafrom Rose Bengal homogeneously incorporated into MSA disks andpolymerized.

FIG. 15 shows the variation of double bond conversion with the residencetime of the poly(ethylene glycol) diacrylate (PEGDA) particles in theapparatus.

FIGS. 16A-D are SEM micrographs of PEGDA particles formed withphotoinitiator concentrations of 1 (A), 1.5 (B), 2 (C), and 4 (D)percent by weight of the monomer.

FIGS. 17A-C are SEM micrographs of PEGDA particles prepared with averageincident light intensities of 3 (A), 4 (B), and 6.25 (C) W/cm².

DETAILED DESCRIPTION OF THE INVENTION

A process for photopolymerizing polymer particles in-situ withantisolvent precipitation is provided. Photopolymerization occurs whensolutions of polymer precursor and solvent are exposed to light ofsufficient power and of a wavelength capable of initiatingpolymerization while being contacted with an antisolvent at reducedtemperature (T_(r)) and pressure (P_(r)). The polymerization may beinitiated by a polymerization initiator activated by light, or aphotosensitive polymer precursor may be used. If a photosensitivepolymer precursor is used, a separate photoinitiator is optional. Thepolymer precursor and solvent solution may be homogeneous, but that isnot required. This type of polymerization results in particles with awide range of morphologies, sizes and physical characteristicsadjustable by changing the process conditions. The polymer particlesproduced by the methods of the invention do not require any furtherprocessing (for example solvent removal) before they may be used.

Not wishing to be bound by any theory, it is believed that the chamberconditions coupled with the antisolvent properties (high diffusivity,low viscosity, and high solvating capacity) facilitates the extractionof the solvent from the solution leaving mostly precursor and initiator(if used). At the same time, these precursor/initiator particles receivephotons from the UV source, initiating the polymerization.

The reduced temperature is the ratio of the operating temperature overthe critical temperature for the antisolvent.$T_{r} = \frac{T_{operating}}{T_{critical}}$

The reduced pressure is the ratio of the operating pressure over thecritical pressure for the antisolvent.$P_{r} = \frac{P_{operating}}{P_{critical}}$

In the methods of the invention, T_(r)=0.7 to 1.3, preferably T_(r)=0.9to 1.1. In the methods of the invention, P_(r)=0.5 to 2, preferably,P_(r)=0.75 to 1.5.

The precursor/initiator/solvent should remain at a temperature such thatthe initiators (if used), precursors and any desired additives are notdegraded to an extent that is unacceptable for the particularapplication. The methods of this invention may be used where theantisolvent is not at reduced pressure and temperature to producepolymer particles, as long as particle formation occurs. The methods ofthe invention may also be used to produce polymer particles when thesolvent or solvent system used is not completely miscible in theantisolvent or antisolvent mixture, or when the precursor is soluble tosome extent in the antisolvent or antisolvent mixture.

Polymers with many different morphologies and physical properties may beproduced using the methods of this invention. The morphology changes inpolymers formed by changing conditions in the PCA experiment have beenstudied (Dixon, D. J. and Johnston, K. P. (1993), “Formation ofMicroporous Polymer Fibers and Oriented Fibrils by Precipitation with aCompressed Fluid Antisolvent,” J. Appl. Polym. Sci. 50:1929-1942).

Polymers with improved mechanical strength, polymer-encapsulatedbioactive materials where the biomaterial has controlled transportproperties through the polymer, and polymers that are capable ofdegrading or remaining substantially intact in a given system, forexample an organism such as a human or other mammal, may be preparedusing the methods of this invention. Nondegradable polymers may beformed using the methods of this invention. Polymer particles may beformed using the methods of the invention for use in many applicationssuch as agricultural controlled release of fertilizer, use as fillers,and other applications. In addition to polymer particles, polymer fibersand porous polymer particles, for example, are achievable by changingone or more process parameters such as the solvent flow rate, polymerprecursor type and functionality, photoinitiator concentration,initiation rate, chamber operating temperature or pressure, among otherparameters.

For example, increasing the concentration of polymer precursor in thesolution increases the diameter of the polymer particles formed. Inaddition, initiating polymerization some distance after the particleshave been formed increases the diameter of the polymer particles formed.The diameter of the polymer particles formed can be increased bymanipulating the nozzle size, increasing the concentration of monomer,increasing the temperature. Polymer fibers as opposed to more sphericalparticles can be formed by using slower flow rates of theprecursor/initiator/solvent.

Copolymers may also be made using this method, as well as bioerodablepolymer particles which can be used, for example, in controlled releaseapplications.

Polymers with crosslinked polymer networks may also be formed using themethods of this invention. In a photoinitiated process, the extent ofcrosslinking may be controlled by, for example, controlling thecarbon-carbon double bond or other reactive functional groupconcentration in the polymer precursor, the intensity of the lightsource, the time of exposure to the light source, and the photoinitiatorconcentration (if present). The time of exposure to the light source, orresidence time, is controlled by the combination of the antisolvent andsolution flow rates. The extent of cross-linking is preferably largeenough to provide gelation of the polymer and prevent agglomeration ofthe particles once they are formed. In general, less reactive polymerswill require more exposure time, higher light intensity, and a higherphotoinitiator concentration. Those skilled in the art can assess therelative reactivities of monomers based on their molecular weights andfunctional groups.

For precursors having C═C functional groups, formation of crosslinksbetween molecules involves conversion of C═C bonds to C—C bonds. Bondswhich have been converted from C—C double bonds can be termed “convertedC—C double bonds.” Similarly, those functional groups containing C—Cdouble bonds which have been converted to C—C single bonds can be termed“converted functional groups”, e.g. converted acrylate or methacrylategroups. The extent of crosslinking can be measured by FTIR analysis ofthe double bond conversion in the polymer particles (referenced todouble bond measurements of the unreacted precursor) or by other methodsas known to the art. Uncertainty in this method of double bondconversion can be ±10%, but is typically ±5%. Similar measures of theextent of crosslinking are known to the art for precursors having othertypes of functional groups.

For polymer networks which swell in solvent, the mesh size of thepolymer network in the particles for a given extent of double bondconversion can be estimated from network mesh sizes for a bulk polymerhaving the same extent of double bond conversion. Mesh sizes for thebulk polymer can be calculated from measurements of the swelling of thebulk polymer as described by Lu and Anseth, (2000), “Release Behavior ofHigh Molecular Weight Solutes from Poly(ethylene glycol)-BasedDegradable Networks”, Macromolecules, pp 2509-2515.

For polymer networks which do not swell in solvent, the mesh size of theparticles can be estimated statistically assuming an ideal network and amonodisperse monomer molecular weight. For macromers having C═Cfunctional groups which are 100% converted to C—C kinetic chains orcrosslinks, the sum of the bond lengths of the repeating unit (excludingside groups) can multiplied by the number of monomer units to obtain thelength of one side of the mesh. The length of the other side of the meshcan be estimated to be the same as the kinetic chain length or C—C bondlength. With an ideal network (100% conversion, no cyclization) the meshsize is the average of these 2 lengths. To calculate for otherconversions, the system can be idealized further. For example, for 50%conversion, it can be estimated that every other monomer unit, therewill be a kinetic chain link, so the length of one side of the mesh canbe estimated as twice the sum of the bond lengths of the startingmonomer multiplied by the number of monomer units. The length of theother side of the mesh can be estimated as the C—C bond length asbefore.

Additives of various sorts may be added to theprecursor/initiator/solvent solution or the antisolvent. These additivesmay include, but are not limited to: plasticizers, coloring agents,encapsulation agents, bioactive materials such as drugs of variouskinds, and other inert or bioactive particles. As used herein,encapsulation efficiency refers to the amount of drug encapsulated intoa quantity of particles (which can be calculated from release data)divided by the amount of drug loaded into an equivalent quantity ofprecursor. The methods of the invention allow improved encapsulationefficiency of additives such as bioactive materials by allowing a widerange of polymer precursors to be used. The polymer precursor can thenbe selected which is compatible with the drug. For example, ahydrophobic bioactive material can be paired with a relativelyhydrophobic polymer.

The degradability of these materials can further be controlled byvarying polymer composition and morphology. This permits tuningdegradation devices to match a desired release rate or release profile.Homogeneous encapsulation of a drug, for example, into polymer particlesin a single manufacturing step is possible using the methods of thisinvention. Changing the size and morphology of the degradable particlesallows control over the dose and duration of the drug delivery.

A variety of embodiments of the invention are possible. For example, onedrug may be encapsulated in a polymer particle using the methods of theinvention. Then, a second polymer precursor, initiator and drug may beused to encapsulate a second drug over the first particle. This willresult in a particle that has two or more different bioactive materialswith different release profiles. This is useful in a variety ofdifferent therapeutic applications.

Methods of determining appropriate dosages for bioactive materials arewell known to one of ordinary skill in the art. Polymer particles andcompositions comprising bioactive materials are administered by methodswell known in the art, or by adapting methods well known in the art.

This invention is useful for other controlled release of materials otherthan drugs. Other applications include controlled release of fragrancesand pesticides. Particles may be made using the methods of the inventionthat release corrosion inhibitors over a specified time. This may beuseful in pipeline applications. Other uses are readily apparent to oneof ordinary skill in the art without undue experimentation.

To circumvent potential problems associated with solubilizing ahydrophilic drug in an organic solvent such as microphase separation andconsequent burst effects, the photopolymerization technique describedherein can be combined with a solubilization technique known ashydrophobic ion-paring (HIP) to form homogeneous solutions of drug,monomer and initiator in an organic solvent and photopolymerizeddrug-encapsulated microparticles. HIP is described in U.S. Pat. Nos.5,981,474 and 5,771,559, hereby incorporated by reference to the extentnot inconsistent with the disclosure herein. HIP is a technique wherebyionic pharmaceutical agents can be directly solubilized in organicsolvents. HIP consists of pairing charges on the molecule withoppositely charged, hydrophobic organic ions, effectively increasing themolecule's solubility in low-dielectric organic solvents. Thephotopolymerization method described herein may be used in combinationwith HIP to encapsulate a therapeutic agent in polymer particles.

Parts per billion residual solvent levels have been obtained for PCAprocessing of linear poly(lactic) acid, in which the particles arewashed with several volumes of CO₂ after processing (Falk and Randolph(1998) Pharmaceutical Research, 15, 8,1233-1237). If the particles ofthe present invention are washed with supercritical fluid such as CO₂after processing, residual solvent levels can be reduced below 1%.

Apparatus for Polymerization Experiments

FIG. 1 illustrates an apparatus of this invention for providing polymerparticles. The apparatus has a chamber (45) with one or more inlets (85,95) that allow substances to pass into chamber (45). In a particularembodiment, antisolvent (20) is connected to optional oxygen scrubber(80) through connecting tubing (50). Oxygen scrubber (80) is connectedto pump (15) through connecting tubing (50). Pump (15) is connected tovalve (5) with connecting tubing (50). Valve (5) is connected to inlet(85) through connecting tubing (50). Inlet (85) allows antisolvent (20)to enter chamber (45). Pump (15) is used to pump solution (10)comprising one or more polymer precursors, one or more initiators andone or more solvents to valve (5) through connecting tubing (70).Solution (10) is pumped to inlet (95) through connecting tubing (70).Inlet (95) allows solution (10) to enter injector (25) inside chamber(45). Light pipe (35) passes light from light source (30) into chamber(45). After polymer particle formation, particles (90) pass out ofchamber (45) to filter (40) and valve (5).

The embodiment described by FIG. 1 illustrates more than one inlet (85and 95). In an alternative embodiment, the antisolvent and solution passinto the chamber through one inlet. The precursor/initiator/solvent mayalso be sprayed into a stream of antisolvent or antisolvent mixture. Inanother alternative embodiment, there are multiple inlets for variouscomponents.

In operation, the following preferred procedure is used. Antisolvent(20) is optionally deoxygenated with oxygen scrubber (80). Antisolvent(20) is pumped with one or more pumps (15) through connecting tubing(50) to valve (5). Antisolvent (20) is then pumped into opticallyaccessible high pressure chamber (45) through inlet (85). The flow rateof antisolvent (20) into chamber (45) is typically about 25 ml/min. Anoptional heating or cooling source (not shown) may be positioned in anysuitable location to provide any necessary heating or cooling to theantisolvent, the chamber, or any part of the apparatus or any componentthereof. Antisolvent (20) is preferably pressurized and heated so thatit is at or above its critical pressure and critical temperature.Chamber (45) is allowed to equilibrate at the desired temperature andpressure (preferably at or above the critical temperature and pressureof the antisolvent). At least one polymer precursor and at least onephotoinitiator are dissolved or suspended in a suitable solvent to formsolution (10). Solution (10) is pressurized to the desired pressure withpump (15). Solution (10) is pumped through connecting tubing (70) tovalve (5) and pumped through connecting tubing (70) to inlet (95).Antisolvent (20) can be co-flowed coaxially with solution (10). Solution(10) passes through inlet (95) into injector (25) into chamber (45). Inone embodiment, injector (25) is a stainless steel tube with a 100 μmopening which injects solution (10) into chamber (45). In anotherembodiment, injector (25) is any type of injector known in the art,including ultrasonic nozzles and laser drilled holes. Many differentnozzles may be used, including a stainless steel capillary tube, aquartz capillary tube, a sonicated nozzle, and a converging divergingnozzle with a premixing chamber. The injector and inlet are not requiredto be separate components. The flow rate of solution (10) throughinjector (25) is typically about 0.1 to about 1 ml/min. Light source(30) provides the necessary photons to initiate photopolymerization at adesired distance (in one embodiment, 2-3 cm) below solution injector(25). In one embodiment, light source (30) is a ultraviolet or visiblelight source at about 800 to about 6300 mW/cm² (30). The light istransferred from light source (30) into chamber (45) using any suitablemeans, including optical fiber (35). Particles (90) may be collected byany suitable means, including the use of filter (40). A 0.2 μm filter isused in one embodiment, but any suitable pore size may be used. The sizeof the pores of the filter needed will depend on the size of theparticles formed and the desired size of particles collected. Particlesmay be transferred from filter (40) through valve (5).

The chamber itself in a preferred embodiment is a 5″×4″×9″ longstainless steel chamber with a volume of 100 ml. Tempered borosilicatewindows (3.5″ long each) are used. The chamber weighs about 50 pounds.Other embodiments of the chamber may be used.

Alternate Apparatus for Continuous Processing

Alternatively, the apparatus may be operated in a continuous mode. Thisis shown in FIG. 2. In this embodiment, injector loop (60) withinjection port (65) such as those used in an HPLC apparatus may be addedto the apparatus to allow processing of multiple solutions or multiplesamples of the same solution without the time consuming pressurizing anddepressurizing cycles that would otherwise be required. In thisembodiment, a flow of solvent (40) is maintained through connectingtubing (70), a flow of antisolvent (20) is maintained through connectingtubing (50), and a solution of polymer precursor/initiator and solventis injected into the solvent flow through injection port (65).

A series of valves and filters may be used to enable particle collectionwithout depressurizing the system. This is also shown in FIG. 2. Theflow path exiting the chamber is split. Particles may be collected onfilter (125) by closing valve (100) and opening valve (105). By closingvalve (105) and opening valve (100), particles may be collected onfilter (120). While particles are being collected on filter (120),filter (125) may be replaced. This way particles may be collectedcontinuously by routing the flow. This allows a continuous, not batch,process to be maintained, and greater amounts of polymer particles maybe produced.

The apparatuses described above are only some of the possibleapparatuses that may be used to carry out the invention. Otherembodiments of the apparatus or components of the apparatus will bereadily apparent to those of ordinary skill in the art. For example, thesolution of polymer precursor(s) and photoinitiator(s) may pass into thechamber through other diameter injectors or injector types other thanthose mentioned specifically. The solution of polymer precursor(s) andphotoinitiator(s) may be optionally heated or cooled in any suitablelocation. Any suitable light source may be used, and any suitable methodof bringing light to the chamber may be used. The light is brought intothe chamber at any desired location. The range of possible modificationsis well known to one of ordinary skill in the art without undueexperimentation.

The invention will be further understood by reference to the followingexamples intended as illustrations, not limitations.

EXAMPLES Preparation of Methacrylated Sebacic Anhydride

The monomer, methacrylated sebacic anhydride (MSA), was prepared bycombining 40 g sebacic acid (Aldrich) with 88 ml methacrylic anhydride(Aldrich) and refluxing for approximately 1 hour. This process, shown inScheme 1, converts the dicarboxylic acid to the anhydride monomer whichis subsequently dissolved in dry methylene chloride (Fisher) andprecipitated in petroleum ether (Aldrich) for purification and recovery(U.S. Pat. No. 4,789,724).

Proton NMR spectroscopy (Varian VXR-300S) was used to verify theexistence of the characteristic methacrylate end-capped ═CH₂ protonsthat give peaks at 5.8 and 6.2 ppm. Infrared spectroscopy (IR) shows thepresence of the methacrylate double bond group at 1635 cm⁻¹ andconfirmed the conversion of the acid groups to the anhydride (Muggli, D.S. et al. (1998) Macromolecules 31:4120-4125). After forming thedimethacrylated monomeric anhydride, the monomer can be oligomerizedthrough a condensation polymerization under vacuum at a temperature of60° C. A ratio of the integrated area of the ═CH₂ proton peaks to theinternal protons in the MSA backbone from the NMR analysis suggests anumber average degree of oligomerization of −6 repeat units.

Initiator Selection

The photoinitiators used in these experiments were Rose Bengal and RoseBengal bis(triethyl ammonium) salt obtained from Aldrich, although otherinitiators may be used. Interestingly, the Rose Bengal and its ammoniumsalt have dramatically different absorbance spectra in methylenechloride. FIG. 3 shows that the large peak at −550 nm seen in the RoseBengal salt is not present in Rose Bengal. Since the chamber window usedin these experiments absorbed wavelengths below 350 nm, visible lightinitiators were used, and the triethyl ammonium Rose Bengal salt, whoseabsorbance spectrum is shown in FIG. 3 was used in these experiments.

Particle Production.

5-20 wt % methacrylated sebacic anhydride was dissolved in methylenechloride along with 2% photoinitiator by monomer weight. The chamber waspressurized with deoxygenated CO₂ by two ISCO compressed gas pumps andallowed to equilibrate to the desired temperature and pressure. Themonomer-solvent solution was then pressurized to the desired pressure bya third ISCO pump. The solution was injected into the pressurizedchamber environment at a constant flow rate (1 ml/min) through thenozzle while the CO₂ flowed at a constant rate of 25 ml/min. A highpowered light source (1-4 W/cm²) (EFOS, Novacure) with a visible filter(350-650 nm) and a fiber optic liquid light guide was used to initiatethe photo-polymerization below the nozzle. A 5 cm Light Line (EFOS) wasused to spread out the beam from the light source to give a longerinitiating time in the chamber.

After spraying and polymerization, the system was allowed to settle forhalf an hour before slow depressurization (˜30 min) at the operatingtemperature. This slow depressurization increased the number ofparticles collected on the scanning electron microscopy (SEM) stubmounted inside the chamber. After depressurization, samples were alsotaken from both the inside of the chamber and the 0.2 μm filter. Theresulting particles were examined using SEM to determine their size andmorphology.

The poly(methacrylated sebacic anhydride) (PMSA) particles were alsoviewed using a fluorescence microscope (data not shown). The Rose Bengalphotoinitiator is a fluorescent dye for the microparticles, with anexcitation peak at 540 nm and an emission band between 550-600 nm, sothe distribution of photoinitiator in the polymerized particles can bevisibly characterized.

Polymerization of the multifunctional anhydride monomers during theparticle processing was confirmed through Fourier transform infrared(FTIR) spectra of the particles compared to the oligomer (FIG. 4). Thepeak at 1635 cm⁻¹ is assigned to the carbon-carbon double bondstretching in MSA and is largely reduced in relative intensity in thespectrum of (PMSA). The reduction of the peak to immeasurable levelsfurther suggests nearly complete reaction possibly due to added mobilityfrom the solvent.

FIG. 5 is a scanning electron microscopy (SEM) photograph of the poly(methacrylated sebacic anhydride) microparticles magnified 2200 times.These particles were sprayed as described above with a nozzle consistingof a 100 μm stainless steel capillary tube and an operating temperatureof 37° C. The particles, although not perfectly spherical, exhibit around or substantially spherical morphology with diameters ranging from5 to 15 μm. This narrow size distribution is an important advantage ofthis processing technique since many applications of polymermicroparticles require a narrow size distribution, especially biomedicalapplications where the body may absorb or reject the particles based ontheir size. The size distribution can also control the release kinetics.

Cloud Point Measurements

Cloud point experiments were performed to measure the solubility of themonomer solution in CO₂ at the initial operating conditions of 8.5 MPaand 37° C. A 3 L view cell was injected with a methylene chloride/MSAsolution and a pump system insured complete mixing. At a constanttemperature of 37° C., the cell was then pressurized with CO₂ using ahand pump up to ˜95 bar. Next, the cell was slowly depressurized and thepressure at which the monomer solution mixture became visibly insolublewas recorded. The process was repeated three times for each MSA solutionconcentration and the cloud point pressures were averaged. These resultsare shown in FIG. 6.

FIG. 6 shows the cloud point pressure for this system, which is farbelow the initial operating pressure implying that the monomer solutionis in a gaseous state at the initial operating conditions of 37° C. and8.5 MPa. If the monomer remains in a gaseous state duringphotopolymerization, this would significantly dilute the monomerconcentration and decrease the rate of polymerization and theprobability of microparticle formation during the exposure time. A rapidpolymerization rate and a low gel point conversion are desired for thissystem because the exposure time after atomization is short (4×10⁻²sec). To circumvent this problem, the operating temperature was loweredto 25° C. such that the operating pressure was below the cloud point.After this adjustment, the monomer polymerized in the chamber andparticles with a high C═C conversion were obtained.

FIG. 7 is an SEM micrograph of PMSA particles magnified 2000 times thatwere precipitated from a 5 wt % MSA solution using an ultrasonicatedatomizing nozzle and an operating temperature of 25° C. Consistentproduct particles with the same size (again 5 to 15 μm) and morphologywere formed at these operating conditions. Although not spherical, theparticle morphology is strikingly similar to the cusped surfaces formedin low pressure PCA with linear polymers. These particle surfacefeatures are speculated to be a result of slow drying or surface-onlypolymerization. A surface-only polymerization would likely result inthese flat, petal-like particles because polymerization would occur onone face of the droplet which might give the “dark” side of the droplettime diffuse into the CO₂ phase. Monomer-solvent solubilities andoperating conditions may be manipulated to obtain solid, sphericalparticles.

FIG. 8 shows SEM micrographs of PMSA particles prepared from 5% (FIG.8A), 10% MSA (FIG. 8B) and 20% MSA (FIG. 8C). The experiment wasperformed at a temperature of 37° C. and a pressure of 85 bar. Noticethe particle size increases with increasing concentration of MSA. Thesame magnification is used for all micrographs of FIG. 8 (500 times).

Triacylate Polymerization

Particles of triacrylate were also formed using the system as describedabove, using the following two compositions:

-   -   A: 10% 1,1,1-trimethylol propane triacrylate, 10% (by monomer        weight) DMPA photoinitiator, 90% methylene chloride.    -   B. 15% triacrylate, 6% (by monomer weight) DMPA photoinitiator,        85% methylene chloride.

The experiments were carried out using carbon dioxide as theantisolvent, using a pressure of 85 bar and a temperature of 37° C.

FIG. 9A shows a SEM micrograph of particles formed using condition Aabove at 10,000 times magnification. FIG. 9B shows a SEM micrograph ofparticles formed using condition B above at 10,200 times magnification.

FIG. 10A shows a SEM micrograph of particles formed using condition Aabove at 5,200 times magnification. FIG. 10B shows a SEM micrograph ofparticles formed using condition B above at 5,100 times magnification.

Copolymer Formation

Copolymers of methacrylated sebacic anhydride (MSA) and polylactic acid(PLA) were formed using the methods of the invention using the followingtwo compositions:

-   -   A: 2.5% MSA, 2.5% PLA, 95% methylene chloride, 20% (by monomer        weight) DMPA photoinitiator.    -   B. 5% MSA, 5% PLA, 90% methylene chloride, 20% (by monomer        weight) DMPA photoinitiator.

The experiments were carried out using carbon dioxide as theantisolvent, using a pressure of 85 bar and a temperature of 37° C.

The SEM of particles from system B are shown in FIG. 11 at 4700 timesmagnification.

Hydrophobic Ion Pairing

The drug tacrine, given to sufferers of Alzheimers disease, has beenencapsulated homogeneously into these microparticles and the releasebehavior of the drug has been studied.

A 0.1 M aqueous solution of dodecyl sulfate, sodium salt (SDS) wasprepared. In addition, a 5-mg/ml aqueous solution of Tacrine was alsoprepared.

9-amino-1,2,3,4-tetrahydroacridine (Tacrine)

Tacrine has one charged site available, and therefore requires a one toone pairing to a surfactant. The appropriate volumes of each solutionwere combined to obtain this stoichiometric ratio of SDS molecules toTacrine molecules. The solution was then mixed vigorously forapproximately 2 minutes. A cloudy solution results from the ion-pairedprecipitate and aqueous phases. Centrifuging the solutions forapproximately 15 minutes at 6000 rpm separates the precipitate from theaqueous phase, allowing the aqueous phase to be removed easily. The wetprecipitate was then dried under vacuum for 24 hrs before use in anyfurther experimentation.

Fluorescence imaging allowed the particles to be examined using adifferent technique since the Rose Bengal initiator is also afluorescent material. FIG. 12 is a fluorescence microscopy image of PMSAmicroparticles encapsulated with tacrine magnified 20 times. Althoughnot perfectly spherical, these particles exhibit a relatively roundshape. From the fluorescence images of the particles, Rose Bengalappears to be evenly distributed on the particle surface. This imagesuggests that the Rose Bengal is dispersed throughout the particles,which may provide many initiation sites for a given particle. Manyinitiation sites could result in a non-uniform surface, where particleformation is dominated by nucleation and growth, rather than byatomization.

Drug Release Protocol

Approximately 2.5 mg of PMSA particles were placed into a 1.5ml-capacity plastic centrifuge vial and filled with phosphate-bufferedsaline (PBS) (pH=7.4) at 37° C. The centrifuge tube was shaken todisperse the particles throughout the solution and placed in a 37° C.temperature bath for 3 minutes. The tube was then immediatelycentrifuged, and the buffer was drawn off and analyzed for drugconcentration. The centrifuge tube was refilled with 37° C. PBS, and thecycle was repeated for about 3 hours. The tubes remained in thetemperature bath for 3 minutes for the first hour, and 5-10 minutes forthe remainder of the time data was collected. UV-Vis spectrophotrometry(Model 8452, Hewlett Packard) was used to determine the concentration oftacrine (absorbance was measured at 322 nm) in experiment samples.

Drug release experiments illustrate the release properties of themicroparticles formed in the photopolymerization PCA process. In allstudies almost all of the particles degraded within 2 hours and thistime scale is consistent with what can be calculated from thedegradation kinetic constant of PMSA investigated by Muggli et al(Muggli, D. S. et al. (1999) J. Biomed. Mater. Res. 46:271-278). A 5-15μm particle would degrade in about an hour, according to the calculatedkinetic constant, but since oligomerized MSA was used, a longerdegradation time should be expected. FIG. 13 shows the release profileof tacrine (absorbance measured at 322 nm (upper data)) and Rose Bengal(absorbance measured at 550 nm (lower data)) from the particles into PBSbuffer. The tacrine release profile follows the curve for surfaceerosion of a sphere as expected. This profile concurs with SEMphotographs that show that the particles have a round shape. The shapeof the release profile of Rose Bengal indicates there is some influenceof diffusion in the release. The Rose Bengal release profile is alsoaffected by photobleaching of particles in room light. This may explainthe lag time at the beginning of the Rose Bengal release in FIG. 13.

Release of Particles from an MSA Matrix

Crosslinked particles containing Rose Bengal were incorporated in a MSAmatrix and polymerized into disks. Rose Bengal was also homogeneouslyincorporated into another set of MSA disks and polymerized. Advantagesof using a particle-polymer composite include multi-mode degradation andrelease possible through the use of different biocompatible polymers anddrugs, ease of control of surface degradation, and in one particularapplication, bone growth is facilitated by the resulting porousstructures.

Disk samples were 0.25 to 0.3 grams, 13 mm diameter and 1.5 mm thick.Disks were placed in 10 ml of PBS buffer at 37° C. to monitor thedegradation.

FIG. 14 shows release behavior for both sets of disks as a function ofthe mass fraction of the disks that had degraded. The absorbance of RoseBengal was measured at 550 nm, and the disks were weighed to monitor thedegradation. FIG. 14 shows a linear relationship between degradation andrelease for both homogeneous and heterogeneous disks.

One particular application of the particles is in bone cements.Particles release drugs over time as the bone cement (formed ofdegradable material) degrades and bone regrows.

Diacrylated Poly(ethylene glycol) (PEGDA) Polymerization

Particles of Poly(ethylene glycol) Diacrylate (PEGDA) were formed usingthe methods of the invention using the PEG1000DA monomer (PEG1 kDA,Monomer-Poymer and Dajac Laboratories, Southhampton, Pa.). Theexperiments were carried out using methylene chloride as the solvent andcarbon dioxide as the antisolvent, using a pressure of 85 bar and atemperature of 35° C. The photoinititator used was2,2-dimethoxy-2-phenlyacetophenone (DMPA, Ciba Geigy, Tarrytown, N.Y.).

Poly(ethyleneglycol) Diacrylate

FIG. 15 shows that the double bond conversion varied with the residencetime of the particles in the apparatus. The solution used in theseexperiments had 25% PEG1000DA and 2% (by monomer weight) DMPA. The lightintensity was 6 W/cm². The value of the double bond conversion indicatesthe extent of cross-linking. The particles corresponding to approx. 20%conversion were agglomerated. The residence time of the particles in theapparatus was varied by manipulating the combination of the antisolventand solution flow rates.

The double bond conversion of the particles was determined through FTIRanalysis compared to that of the PEG1000DA monomer. Particles werecombined with mineral oil and crushed uniformly using a mortar andpestle to create a smooth paste of oil and crushed particles. A spectrum(64 scans averaged) was acquired in the mid-IR region of the resultingpaste sandwiched between two KBR crystals. The same technique was usedto make samples of the PEG1000DA monomer. Fractional conversion wascalculated by subtracting the area of the carbon-carbon double bond peakof the reacted sample from that of the unreacted monomer and thendividing by the peak area of the unreacted monomer.

Experiments were also performed to assess the influence of the amount ofphotoinitiator on the process. FIGS. 16A-D, respectively, show SEMmicrographs of the particles formed with photoinitiator concentrationsof 1, 1.5, 2, and 4 percent by weight of the monomer (25 wt % monomer, 6W/cm² incident light intensity). The solution flow rate was 1 ml/min. Asshown in FIGS. 16B and 16C, The 1.5 and 2 wt % initiator samples formedvery smooth, spherical particles with diameters in the range 0.5 to 50microns. In contrast, the samples processed with 1 and 4 wt % initiatorgenerally look like agglomerated particles with little uniformity instructure, as is shown in FIGS. 16A and 16D. In the case of the 1 wt %DMPA photoinitiator sample, the rate of polymerization was likelyinsufficient to produces significant conversion that would preventparticle agglomeration. In the case of the 4 wt % DMPA photoinitiatorsample, an excess of initiator likely led to decreased double bondconversion (Lovell, L. G.; Berchtold, K. A.; Elliott, J. E.; Lu, H.;Bowman, C. N. Polym. Adv. Technol. 2001, 12, 335-345. and Kloosterboer,J. Adv. Poly. Sci. 1988, 84, 1-61).

The effect of varying light intensity during the process was alsoexamined. FIGS. 17A-C, respectively, show particles prepared withaverage incident light intensities of 3, 4, and 6.25 W/cm². The lightintensities were measured using an EFOS Novacure Radiometer,Mississauga, Ontario, Canada. Other experimental conditions were 25 wt %monomer and 2 wt % photoinitiator relative to monomer. As shown in FIG.17A, samples prepared at the low light intensity (3 W/cm²) show littleto no structure, judging from the large agglomerated masses and minimalevidence of particle formation. The medium intensity experiment (4W/cm²) exhibits an increased amount of particle formation but stillsignificant numbers of aggregates (FIG. 17B). The high intensityexperiment (6.25 W/cm²) produced nicely formed spheres in size rangesfrom 1 to 50 microns (FIG. 17C). These results suggest that greaterlight intensity is more effective in producing crosslinked sphericalparticles in the limited time that the particles have to polymerizebefore they will interact with other particles in the high-pressurechamber due to fluid mixing that occurs in the process.

Calculation of Mesh Sizes for PEGDA Networks

The mesh sizes were calculated statistically assuming an ideal networkand a monodisperse monomer molecular weight. For the starting macromer,PEG (—C—C—O—)_(n), the PEG based chain had a bond length of 4.34Angstroms for 2 C—O bonds at 1.4 angstroms each and one C—C bond at 1.54Angstroms. For 100% double bond conversion, the length one side of themesh was estimated as n*4.34 Angstroms. The length of the other side ofthe mesh was estimated to be the same as the kinetic chain length or C—Cbond length. For an ideal network (100% conversion, no cyclization) themesh size was estimated as the average of these 2 lengths.

For example, for 50% conversion, it was estimated that there will be akinetic chain link every other monomer unit. The length of one side ofthe mesh was estimated as twice the sum of the bond lengths of thestarting monomer multiplied by the number of monomer units. The lengthof the other side of the mesh can be estimated as the C—C bond length asbefore.

For a completely reacted system (100% double bond conversion) thecalculated mesh sizes were 12.0 Angstroms for PEG200DA, 30 Angstroms forPEG 600DA, and 50.4 Angstroms for PEG1000DA. For a partially reactedsystem with 50% double bond conversion the calculated mesh sizes were23.3 Angstroms for PEG200DA, 59.4 Angstroms for PEG 600DA, and 100.0Angstroms for PEG1000DA.

Encapsulation Efficiencies

The encapsulation efficiency measured for tacrine in MSA was 92±4%, thatin PEG10000DA was 31±7%, that for PEG600DA was 26±7%, and that forPEG200DA was 85±24%.

Although the description above contains many specificities, these shouldnot be construed as limiting the scope of the invention but as merelyproviding illustrations of some of the presently preferred embodimentsof the invention. For example, antisolvents other than carbon dioxidemay be used. Other embodiments and uses are readily apparent to one ofordinary skill in the art without undue experimentation. Thus, the scopeof the invention should be determined by the appended claims and theirlegal equivalents, rather than by the examples given. All referencescited herein are hereby incorporated by reference to the extent notinconsistent with the disclosure herewith.

1. Crosslinked polymer particles, wherein the particles are between 0.1and 200 microns in diameter and have a network mesh size between about10 and about 500 Angstroms.
 2. The particles of claim 1, wherein thepolymer is selected from the group consisting of: vinyl acetates, vinylpyrrolidones, vinyl ethers, olefins, styrenes, vinyl chlorides,ethylenes, acrylates, methacrylates, nitriles, acrylamides, maleates,epoxies, epoxides, lactones, ethylene oxides, ethylene glycols,ethyloxazolines, amino acids, saccharides, proteins, anhydrides, amides,carbonates, phenylene oxides, acetals, sulfones, phenylene sulfides,esters, fluoropolymers, imides, amide-imides, etherimides, ionomers,aryletherketones, amines, phenols, acids, benzenes, cinnamates, azoles,silanes, chlorides, and epoxides.
 3. The particles of claim 1, whereinthe polymer comprises a plurality of converted carbon-carbon double bondfunctional groups.
 4. The particles of claim 3, wherein the conversionof the carbon-carbon double bonds is between about 20% and about 100%.5. The particles of claim 3, wherein the conversion of the carbon-carbondouble bonds is between about 70% and about 100%.
 6. The particles ofclaim 1, wherein the polymer comprises a plurality of converted acrylategroups.
 7. The particles of claim 1, wherein the polymer ispoly(ethylene glycol) (PEG) diacrylate.
 8. The particles of claim 1,wherein the polymer comprises a plurality of converted methacrylategroups.
 9. The particles of claim 1, wherein the polymer ismethacrlyated sebacic anhydride (MSA)
 10. The particles of claim 1,wherein the polymer is a copolymer.
 11. The particles of claim 1,wherein the polymer is a copoly(PEG-b-D,L PLA) diacrylate.
 12. Theparticles of claim 1, wherein the polymer is biodegradable.
 13. Theparticles of claim 1, wherein the particles comprise less than about 1%of residual solvent.
 14. The particles of claim 1, wherein the polymeris a functionalized polymer having at least one unreacted reactive groupcomprising a carbon-carbon double bond.
 15. The particles of claim 3,wherein the reactive group is selected from the group consisting ofacrylates, methacrylates, alkenes and alkynes.
 16. Crosslinked polymerparticles wherein the particles are between 0.1 and 200 microns indiameter, the polymer comprises a plurality of converted carbon-carbondouble bond functional groups, and the conversion of the carbon-carbondouble bonds is between about 20% and about 100%.
 17. Crosslinkedpolymer particles wherein a bioactive material is encapsulated withinthe polymer.
 18. The particles of claim 17 wherein the encapsulationefficiency of the bioactive material is above about 60%.
 19. Theparticles of claim 17 wherein the polymer comprises a plurality ofconverted acrylate groups.
 20. The particles of claim 17 wherein thepolymer comprises a plurality of converted methacrylate groups.
 21. Theparticles of claim 17 wherein the polymer is selected from the groupconsisting of: poly(ethylene glycol) (PEG) diacrylate, methacrylatedsebacic anhydride (MLA), and copoly(PEG-b-D,L-PLA) diacrylate.
 22. Theparticles of claim 17 wherein the bioactive material has a molecularweight less than about 1000 Da.
 23. The particles of claim 17 whereinthe bioactive material is selected from the group consisting of:tacrine, erythromycin, erythromycin estolate, and erythromycinethylsuccinate.